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EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION 

COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN 

THE FEDERAL REGISTER 

EPA Biopesticides and Pollution Prevention Division contact: Driss Benmhend, 703-

308-9525, benmhend.driss@epa.gov.

INSTRUCTIONS: Please utilize this outline in preparing the pesticide petition. In 

cases where the outline element does not apply, please insert “NA-Remove” and 

maintain the outline. Please do not change the margins, font, or format in your 

pesticide petition. Simply replace the instructions that appear in green, i.e., “[insert 

company name],” with the information specific to your action.

SUBMISSION: E-mail the completed template to: hollis.linda@epa.gov.

TEMPLATE:

DSM Food Specialties B.V.

Petition number to be assigned.

EPA has received a pesticide petition (To be assigned) from DSM Food 

Specialties B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands,

proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act 

(FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180.

(Options (pick one)

NA-Remove: 1. by establishing a tolerance for residues of

2. to establish an exemption from the requirement of a tolerance for

NA-Remove: 3. to establish an amendment/expansion of an existing tolerance 

exemption for the

(Options (pick one)

NA-Remove: 1. microbial pesticide [insert name of active ingredient]

2. biochemical pesticide (6,11,28-Trioxatricyclo[22.3.1.05,7]octacosa8,14,16,18,20-pentaene-25-carboxylic acid, 22-[(3-amino-3,6-dideoxy--D-
2

mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-, 

(1R,3S,5R,7R,8E,12R,14E,16E,18E,20E,22R,24S,25R,26S)-, CAS Registry No. 7681-

93-8.

NA-Remove: 3. plant-pesticide [insert name of active ingredient] in or on 

[insert commodity].

Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, DSM Food 

Specialties has submitted the following summary of information, data, and arguments in 

support of their pesticide petition. This summary was prepared by DSM Food Specialties 

and EPA has not fully evaluated the merits of the pesticide petition. The summary may

have been edited by EPA if the terminology used was unclear, the summary contained 

extraneous material, or the summary unintentionally made the reader conclude that the 

findings reflected EPA’s position and not the position of the petitioner.

I. DSM Food Specialties Petition Summary

Petition number to be assigned.

A. Product Name and Proposed Use Practices

The Natamycin active ingredient (AI), a polyene macrolide antimycotic 

biochemical substance, was discovered in a soil sample obtained near 

Pietermaritzburg in the state of Natal in South Africa. This biochemical substance 

was found to have broad, potent antifungal properties, but no antibacterial activity. 

The lack of antibacterial properties is a distinctive advantage for a fungicidal 

pesticide because then there is no basis for concern over development of resistance 

by human pathogenic bacteria. 

During mushroom production, Natamycin will be used in a broad spectrum 

liquid fungicidal formulation containing approximately 10% by weight of the 

technical grade active ingredient (TGAI) as a stable aqueous suspension of crystals. 

This product, Natamycin L, has anti-germinating properties and is designed for the 

control of moulds and yeasts in the growth media of mushrooms. Natamycin L can 

be applied to the growth substrate up to four times during a typical production cycle

for mushrooms. Pre-harvest intervals from a few days to more than 20 days .

B. Product Identity/Chemistry

1. Identity of the pesticide and corresponding residues. 

Natamycin. No residues other than Natamycin were found.

2. Magnitude of residues at the time of harvest and method used to determine the 

residue. The proposed use in the present application is for the control of Verticillum 
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(Dry Bubble) disease and other mushroom parasites in growing mushrooms. The 

application produces Natamycin residue levels in mushrooms in the 0.15-0.25 ppm 

range. After rinsing, the residues for the same mushrooms decrease to 0.022 - 0.076 

ppm. No metabolites nor degradation products of Natamycin were found. LCMS/MS methods were used for the analyses. Quantification of Natamycin residues 

was accomplished relative to an external calibration curve. The limit of 

Quantitation (LOQ) for the Natamycin residue method was 0.01 ppm in 

mushrooms. The limit of detection was assigned as the lowest concentration within 

the calibration curve or 0.25ng/ml. The analytical method to detect and measure 

natamycin residues and residues of metabolites, if they were present, was fully 

validated.

3. A statement of why an analytical method of detecting and measuring the levels of the 

pesticide residue are not needed. A worst-case estimate of the Natamycin exposure in 

the diet were calculated using the following: 1) Projected (and increased) mushroom 

intake several years into the future; 2) Use of the mushroom consumption data of 

the most highly exposed population group; and 3) Use of the maximum residue 

found in the crop field trials in any treated mushroom, rather than the most 

probable residue. On this basis, calculations for exposure to Natamycin through 

consumption of treated mushrooms yielded a Margin of Exposure (MOE) of 

approximately one million. 

This very large worst-case MOE clearly establishes the safety of Natamycin 

for the proposed use on mushrooms. Additionally, under normal conditions 

mushrooms are rinsed prior to consuming and the actual MOE will be substantially 

greater than a million. 

Because the MOE is so large even when worst-case assumptions of exposure 

are made, an exemption from tolerance rather than a numeric tolerance is 

appropriate, and an analytical method for detecting and measuring pesticide 

residues in mushrooms is not needed.

C. Mammalian Toxicological Profile

Acute Toxicity Profile

ACUTE ORAL TOXICITY – Rat Toxicity Category III

The test substance, Natamycin, was evaluated for its acute oral toxicity potential in 

female albino rats when administered as a gavage dose at a level of 2000 mg/kg. The 

study was terminated following the stopping rules of this procedure. One of five 

animals died during the study. There were no clinical signs of toxicity in survivors 

during the study; polyuria was observed in the animal that died on test. There was 

no effect on body weight gain in animals surviving to termination. Abnormal 

necropsy findings occurred only in the animal that died on test, and pertained to 

anogenital area, lungs and contents of the intestines. The acute oral LD50 was 

estimated to be greater than 2000 mg/kg.
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ACUTE DERMAL TOXICITY – Rat Toxicity Category IV

The test substance, Natamycin, was evaluated for its dermal toxicity potential and 

relative skin irritancy when a single dose moistened with 1.0 mL of deionized 

water/g test substance, at a level of 5050 mg/kg, was applied to the intact skin of 

albino rats. No mortality occurred during the study. There were no clinical signs of 

toxicity or signs of dermal irritation at any time throughout the study. There was no 

effect on body weight gain, with the exception of one animal that failed to gain 

weight during the first week. The gross necropsy conducted at termination of the 

study revealed no observable abnormalities. The estimated LD50, as indicated by 

the data, was determined to be greater than 5050 mg/kg.

ACUTE INHALATION TOXICITY – Rat Toxicity Category IV

The test substance, Natamycin, was evaluated for its acute inhalation toxicity 

potential in albino rats. Five males and five females were exposed for four hours to 

an aerosol generated from the undiluted test substance (fine powder) at a level of 

2.39 mg/L. There was no mortality during the study. Clinical signs included activity 

decrease and piloerection, which were no longer evident by Day 3. Body weights 

were unaffected by exposure. The gross necropsy revealed no observable 

abnormalities. As indicated by the data, the acute inhalation LC50 is greater than 

2.39 mg/L.

EYE IRRITATION – Rabbit Toxicity Category IV

An acute eye irritation study was conducted on three albino rabbits using test 

substance Natamycin. The test substance, 0.1 mL by volume (43.7 mg), was placed 

into the conjunctival sac of the right eye of each animal selected for testing. All 

treated eyes were washed with room temperature deionized water for one minute 

immediately after recording the 24-hour observation. 

A maximum average irritation score of 62.0, severely irritating, was obtained 1 hour 

after treatment, but there were no positive effects exhibited in any eyes at 24 hours 

after treatment. Hence, the initial irritation score could not be maintained and 

Natamycin TGAI was assigned to Toxicity Category IV.

SKIN IRRTIATION – Rabbit Toxicity Category IV

A primary dermal irritation study was conducted on three albino rabbits using test 

substance Natamycin. There was one intact test site per animal. Each test site was 

treated with 500 mg of test substance moistened with 0.5 mL of deionized water and 

covered with a semi-permeable dressing. The test substance was maintained in 

contact with the skin for 4 hours. Observations for dermal irritation and defects 

were made at 1, 24, 48 and 72 hours after removal of the dressings.
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Based on the PII of 0.1, the test substance is rated slightly irritating. Based on the 

scores at the 72- hour observation only, the test substance is assigned to Toxicity 

Category IV.

SKIN SENSITIZATION – Mice Not a Sensitizer

A skin sensitization study was conducted on 3 groups of 5 female mice to determine 

if test substance Natamycin possesses a significant potential to cause skin 

sensitization. Five females were assigned to each of three groups, designated Groups 

I - III. The test groups were treated with an appropriate dilution (25% or 50%) in 

propylene glycol vehicle, or undiluted test substance. Each animal received 25 μL to 

the dorsum of each ear. The animals were treated once daily for three days. After a 

two-day rest period, all animals were injected with tritiated methyl-thymidine in the 

tail vein. Five hours later, the animals were sacrificed, and the draining auricular 

lymph nodes removed and prepared for cell suspension and scintillation counting. A 

vehicle control group of five females was run concurrently, treated in the same 

manner with vehicle only instead of test substance or dilution. A positive control 

group of five females was also run concurrently, treated with 90% alphahexylcinnamaldehyde in acetone:olive oil.

The test substance produced a stimulation index of < 3 in all groups of test animals, 

and is not therefore considered a sensitizer (defined as producing a positive 

response).

Genotoxicity.

NATAMYCIN was tested in the Salmonella typhimurium reverse mutation assay 

with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, 

TA100 and TA98) and in the Escherichia coli reverse mutation assay with a 

tryptophan-requiring strain of Escherichia coli WP2uvrA . The test was performed 

in two independent experiments in the presence and absence of S9-mix (Aroclor1254 induced rat liver S9-mix). Based on the results of this study it is concluded that 

NATAMYCIN is not mutagenic in the Salmonella typhimurium reverse mutation 

assay and in the Escherichia coil reverse mutation assay.

NATAMYCIN (88.9% purity) was tested to determine the number of chromosome 

aberrations in cultured peripheral human lymphocytes in the presence and absence 

of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) . 

NATAMYCIN did not induce a statistically significant or biologically relevant 

increase in the number of cells with chromosome aberrations in the absence and in 

the presence of S9-mix, in two independently repeated experiments. NATAMYCIN 

is not clastogenic in human lymphocytes under the experimental conditions 

described in this report.

Reproductive and Developmental Toxicity
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Natamycin (as Delvocid 5%) was administered daily during days 6-18 of gestation 

by oral intubation to groups of 15 to 19 female Dutch Belted rabbits at 

concentrations of 0, 5, 15, or 50 mg/kg-day . The administration of up to 50 mg/kg 

(body weight)-day of test material for 13 consecutive days had no clearly discernible 

effect on nidation or on maternal or fetal survival. The number of abnormalities 

seen in either soft or skeletal tissues of the test groups did not differ from the 

number occurring spontaneously in the sham-treated controls. From the results 

presented in this report the No Observed Adverse Effect Level (NOAEL) for the 

teratology part of the study was established to be 50 mg/kg/day.

Subchronic Toxicity

In a 90 day study, Natamycin was administered daily via the diet to groups of 20 

Wistar rats (10 males and 10 females) at concentrations of 0, 125, 500 and 2000 

ppm. Dietary exposure to Natamycin resulted in no treatment related findings in 

the 125 mg/kg/day and 500 mg/kg/day dose groups. The 2000 mg/kg/day dose group 

had reduced body weights (males and females), increased alanine aminotransferase 

activity in individual males, increased urea and inorganic phosphate values (males 

and females) and potassium values (males), and reduced cholesterol and total 

protein values (females). From the results presented in this report the No Observed 

Adverse Effect Level (NOAEL) was established to be 500 ppm Natamycin, 

equivalent to approximately 38 and 43 mg/kg/day (corrected for food scatter) for 

males and females respectively.

Toxicology Profile -- Existing U.S. Government findings of safety

 

Natamycin (pimaricin) has been approved by FDA in 21 CFR Part 172, Sec. 172.155 

for use as a direct food additive for use on cheese. “The additive may be applied on 

cheese, as an antimycotic, in amounts not to exceed 20 milligrams per kilogram (20 

parts per million) in the finished product . . .”. Natamycin as a 5% ophthalmic 

suspension is approved as an antifungal drug for topical ophthalmic administration 

. In 21 CFR Part 573, Sec. 573.685 FDA has approved Natamycin for use in broiler 

chicken feeds at up to 10 grams per pound of food .

Toxicology Profile – International Findings of Safety

The Food and Agricultural Organization of the United Nations (FAO) and the 

World Health Organization (WHO) through the Joint FAO/WHO Expert 

Committee on Food Additives (JECFA), evaluated Natamycin, most recently in 

2002, for safety as a direct food additive . The Committee concluded that “Although 

use of Natamycin as an antifungal agent in food may result in exposure of the 

endogenous flora to trace quantities of antimicrobial residues, bacteria in the 

human gastrointestinal tract are not affected by polyenes, and the Committee 

concluded that disruption of the colonization barrier is not a concern. Fungi are 

found in much smaller amounts than bacteria in the human gastrointestinal tract, 

and the negative results in studies of acquired resistance indicate that selection of 
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Natamycin-resistant fungi is not an issue. The Committee confirmed the previously 

established ADI of 0–0.3 mg/kg bw for Natamycin, which was based on observations 

of gastrointestinal effects in humans.”

The Animal Metabolism of Natamycin

As described in the WHO evaluation , studies have been done to determine the fate 

of Natamycin in rats and dogs. In rats, a series of studies showed that the material 

was not absorbed and was eliminated in the feces and urine. The results of the 

autoradiographic study indicate that Natamycin is minimally absorbed into the 

bloodstream and excreted almost entirely in the feces. Other experiments gave 

similar results. In the stomach and small intestine, Natamycin was mostly 

untransformed, as indicated by thin-layer chromatography. Most degradation took 

place in the large intestine. The degradation products were more hydrophobic than 

Natamycin and were found from about 4 h after treatment. Most of the dose of 10 

mg/kg was degraded about 8 h after treatment, suggesting that elimination is 

relatively rapid. Biotransformation was attributed to the bacterial flora in the 

caecum and small intestine. Overall, no more than 5–7% of the total radioactive 

dose was absorbed after oral administration of [14C]Natamycin, and approximately 

90% of the administered compound passed through the gastrointestinal tract 

without resorption and was eliminated in the faeces . In dogs, after oral 

administration, most of the radiolabel was eliminated in the faeces within 24 h, with 

less than 4% of the total dose in urine. The WHO assessment concluded that there 

was very little data on humans, but one study had shown that “No Natamycin (< 1 

μg/ml) could be detected in the blood after ingestion of 500 mg by human subjects”, 

indicating that it was unlikely to be absorbed by humans.

D. Aggregate Exposure

1. Dietary exposure. Natamycin is currently permitted as an FDA food 

additive, and is approved as an antimycotic for use in the finished cheese at a level 

of 20 ppm. (See 21 CFR § 172.155). The lowest NOEL for Natamycin is 25 mg/kg 

bw/day or approximately 1.75 g/person per day. The proposed use in the present 

application is for the control of Verticillum disease and other mushroom fungal 

parasites in growing mushrooms. The application produces Natamycin residue

levels in mushrooms in the 0.15-0.25 ppm range. After rinsing, the residues for the 

same mushrooms decrease to 0.022 - 0.076 -ppm. 

i. Food. The current U.S. annual per capita mushroom consumption is 4.00 

pounds. The projected maximum, mushroom consumption for the current decade to 

the year 2012 is 4.86 pounds. The maximum daily intake of Natamycin residues for 

the proposed use of Natamycin is conservatively estimated to be 32 ng/kg.bw/day 

resulting in a Margin of Exposure of approximately one million. 
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ii. Drinking water. All uses of Natamycin L will be indoor, and natamycin has 

only very limited (~30-40 ppm at neutral pH) solubility in water. Hence, exposure 

through drinking water is not expected.

2. Non-dietary exposure. The only non-dietary exposures from pesticidal uses 

of the Natamycin will be occupational, i.e., commercial applicator/mixer loader 

exposures. Occupational exposures are not included under the Federal Food, Drug 

and Cosmetic Act (FFDCA) in the assessment of aggregate exposures for the 

purpose of establishing tolerances and exemptions from tolerance.

E. Cumulative Effects In assessing the cumulative effects of Natamycin it should be 

recognized that it is one of a class of polyene antimycotics naturally present in the 

environment. Others of the class are also typically obtained from some species of 

Streptomyces bacteria. The amount of material likely to be present as residues of 

Natamycin on mushrooms results in so small an incremental exposure as to be 

negligible.

F. Safety Determination

1. U.S. population. As discussed above, when expressed in pounds per day, 

per capita mushroom use is highest among men and women aged 20-39, and lowest 

for children under the age of 12. When expressed on a kilogram body weight basis, 

the intakes for all individuals are all within a factor of 2, with the 29-39 age group 

still being slightly higher consumers, particularly in females.

2. Infants and children. On a unit bodyweight basis, there is no significant 

difference in children’s consumption from that of the average adult.

G. Effects on the Immune and Endocrine Systems

Because Natamycin is minimally absorbed into the bloodstream and rapidly 

excreted almost entirely in the feces with minimal degradation, effects on the 

immune and endocrine systems are not expected. The limited biotransformation 

found was attributed to the bacterial flora in the caecum and small intestine, and 

the degradation products were more hydrophobic than Natamycin.

H. Existing Tolerances

There are no existing U.S. tolerances for Natamycin as a pesticide.

I. International Tolerances

No international tolerances exist for use of Natamycin as a pesticide.
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